1. Field of the Invention
This invention relates to the long-term storage of biological materials and reagents. In particular, it relates to compositions for glassy and porous room temperature stable biological reagent semi-spheres and methods for making the same.
2. Background of the Art
Few biologically active materials are sufficiently stable so that they can be isolated, purified, and then stored in solution at room temperature. Typically, biological reagents are stored in a glycerol solution which is maintained at temperatures of 4.degree. C., -20.degree. C., or -70.degree. C. They may be stored in bulk and then combined with other reagents before use.
Biological reagents are sometimes also provided in dried form to increase their storage stability. Furthermore, in preparing reagents for convenient and efficient testing of biological samples, it is frequently important to obtain dry chemical blends in uniform, discreet amounts. These reagents must be efficiently and economically prepared in small, precisely measured quantities. Current technology for producing dry biological reagent compositions involves procedures such as dry-blending, spray-drying, freeze-drying, fluidized bed drying, and/or cryogenic freezing. All these procedures, however, have limitations and drawbacks.
In dry-blending technology, it is often difficult to obtain homogeneous blends of chemicals due to their different densities. Furthermore, homogeneity is especially difficult to achieve when very small amounts of ingredients are mixed with large amounts of others. Even if homogeneity is achieved, it is most difficult to reproducibly dispense small amounts of the blended biological chemicals.
Spray-drying technology provides more homogeneous blends of chemicals because the reagents are first dissolved in solution. See U.S. Pat. No. 4,712,310. For an example of the use of spray-drying along with fluidized bed technology see A. Kassem, et al., 40 Pharm. Ind. 396-399 (1978) and M. Brophy, et al., 33 J. Pharm. Pharmacol. 495-499 (1981). With spray-drying, however, it is difficult to dispense precise amounts of blended chemicals. To overcome this drawback, the resulting particles are usually reprocessed by agglomeration to obtain uniform particle sizes such as tablets. However, the agglomerated particles are generally less soluble than the original spray-dried particles or powders. Also, these procedures sometimes use fluorocarbon cryogenic solutions which can be hazardous to the environment. The disclosure of the above articles and patents, and of all other articles and patents recited herein, are incorporated by reference as if fully set forth herein.
Fluid bed technology relies upon spraying a liquid reagent blend onto a particle and drying the composition to obtain a particle coated with blended reagents. For examples of this technology, ..See, for example, U.S. Pat. No. 4,820,627 and I. Ghebre-Sellassie, et al., 11 Drug Devel. and Indus. Pharm. 1523-1541 (1985). However, using fluid bed technology, it is difficult to obtain uniformly sized particles and to produce a uniform coating.
Another method for stabilizing biologicals is freeze-drying. For examples of various applications of freeze-drying technology see, for example, G. Orndorff, et al., 10 Cryobiology 475-487 (1973); A. MacKenzi, in Freeze-drying and Advanced Food Technology, S. Goldblith, et al. (Eds.), Academic Press, London (1975); U.S. Pat. No. 3,721,725; U.S. Pat. No. 4,134,943; U.S. Pat. No. 4,762,857; U.S. Pat. No. 4,806,343; U.S. Pat. No. 4,897,353; and Japanese patent Application No. 0129102. One drawback to the freeze-drying is the use of fluorocarbon refrigerants which may be dangerous to the environment. Furthermore, freeze-drying is costly in capital and energy, and also suffers from technical disadvantages because of its irreproducibility. Moreover, this process can only be applied to freeze stable biologicals.
Another method of stabilizing biologicals is by air-drying biological reagent compositions. For examples of air-drying of biological compositions using disaccharides as stabilizers see J. Carpenter, et al., 24 Cryobiology 455-464 (1987) and U.S. Pat. No. 4,891,319. Some problems with air drying processes are that the dried product is not in readily dispensable form. Also, the biological reagents must be stable at or above the temperature of the drying process.
One specialized process using freeze-drying technology is the formation of droplets or spheres which are contacted with a cryogenic liquid and then freeze-dried. For examples see U.S. Pat. No. 3,932,943; U.S. Pat. No. 4,780,285; U.S. Pat. No. 4,848,094; U.S. Pat. No. 4,863,856; and PCT Application W093/04195. One drawback of this technology is that the reagent spheres are fragile and tend to disintegrate.
One type of carrier or filler which has been used to stabilize biological reagents are glass forming filler materials. The biological reagent solutions are incorporated into the glass forming filler materials (which are water soluble or a water-swellable substance). They are then dried to produce a glassy composition which immobilizes and stabilizes the biological reagent. For examples of glass forming filler materials for stabilizing biological reagents see, for example, F. Franks, 12 Bio-Technology 253 (1994); U.S. Pat. No. 5,098,893; U.S. Pat. No. 5,200,399; and U.S. Pat. No. 5,240,843.
Carbohydrates such as glucose, sucrose, maltose or maltotriose are an important group of glass forming substances. Other polyhydroxy compounds can be used such carbohydrate derivatives like sorbitol and chemically modified carbohydrates. Another important class of glass forming substances are synthetic polymers such as polyvinyl pyrrolidone, polyacrylamide, or polyethyleneimine.
Further examples of glass forming substances include sugar copolymers such as those sold by Pharmacia under the registered trademark FICOLL. FICOLL polymer is disclosed in U.S. Pat. No. 3,300,474 which describes the materials as having molecular weights of 5,000 to 1,000,000 and as containing sucrose residues linked through ether bridges to bifunctional groups. Such groups may be an alkylene of 2, 3 or more carbon atoms but not normally more than 10 carbon atoms. The bifunctional groups serve to connect sugar residues together. These polymers may, for example, be made by reaction of sugar with a halohydrin or bis-epoxy compound. A glass forming filler material is typically defined as an undercooled liquid with a very high viscosity, that is to say at least 10.sup.13 Pa.times.s, probably 10.sup.14 Pa.times.s or more.
One drawback of the aforementioned references is that normally the stabilized and glassified biological materials are ground into powders, compounded into tablets, or maintained in a thin glassy film in a container like a microcentrifuge tube. This type of packaging is generally inconvenient because dosages of a powdered material are difficult to measure, compounded tablets are slow to dissolve, and excessive sample is needed to dissolve a thin glassy film disposed in a microcentrifuge tube.
Numerous methods to make and use compositions of glassy immobilized biological materials have been tried. One system mentioned above is utilizing a thin glassy film dried and disposed in a container suitable to the final user, such as a microcentrifuge tube. However, attempts have been made to reduce the associated packaging cost by converting the glassy format to a tablet, pellet, or sphere which could be packaged in bulk containers ready for individual use. Various techniques have been tried such as a tablet press, centrifugal granulator, and fluid bed coating and vacuum drying droplets on a flat surface. Each process resulted in limited success.
The tablet press makes easily handled pills but the pills dissolve slowly. The use of tabletting excipients designed to increase dissolution speed interfere with enzyme activity. The centrifugal granulator makes spheres but the size distribution was too large to make individual dispensing practical and all enzyme activity was lost during the drying step. Fluid bed coating made spheres with good size distribution, and activity, but poor solubility. Vacuum drying droplets with a standard low viscosity solution, such as 8%-20% solids, produced flat fragile disks which dissolved slowly.
Glassy substances are also used as hard coatings for candies and pharmaceuticals. Examples of these are found in U.S. Pat. No. 3,456,050; U.S. Pat. No. 4,372,942; U.S. Pat. No. 4,423,086; U.S. Pat. No. 4,559,298; U.S. Pat. No. 4,753,790; U.S. Pat. No. 4,898,781; U.S. Pat. No. 4,997,654; PCT Publication No. W086/00336; and European Patent Application No. 0 252 750.
Accordingly, there is a need for a glassy format biological reagent which possesses excellent water solubility and dissolution rate, possesses a porous structure to assist with dissolution, avoids the typically fragile nature of the reagent spheres disclosed in PCT Publication No. W093/04195 (supra), can be made by a method which allows manipulation of droplet size characteristics, and which can be dispensed from a holder such as that disclosed in U.S. Pat. No. 4,780,285.